Fli1 Is a Negative Regulator of Estrogen Receptor α in Dermal Fibroblasts  Tomoyasu Hattori, Lukasz Stawski, Sashidhar S. Nakerakanti, Maria Trojanowska  Journal of Investigative Dermatology  Volume 131, Issue 7, Pages 1469-1476 (July 2011) DOI: 10.1038/jid.2011.63 Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Estrogen receptor α (ERα) mRNA and protein expression is upregulated by transforming growth factor-β (TGF-β) in human dermal fibroblasts. (a) Reverse transcription-PCR (RT-PCR) analysis of ERα in fibroblasts. (b) Western blot analysis of ERα protein in fibroblasts (50μg) and MCF-7 cells (30μg). The blots were reprobed with anti-β-actin antibody. (c) Western blot of ERα protein determined in serum-starved dermal fibroblasts treated with transforming growth factor-β1 (TGF-β1; 2ngml−1) for 24hours. Representative data of three independent experiments are shown with quantitative representation obtained by densitometric analysis (lower left panel). ERα mRNA levels were determined by quantitative RT-PCR (lower right panel). Values are normalized relative to control (arbitrarily set as 1). Means±SD of three independent experiments is shown. *P<0.05 (Student's t-test) versus control. Journal of Investigative Dermatology 2011 131, 1469-1476DOI: (10.1038/jid.2011.63) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Friend leukemia integration factor 1 (Fli1) downregulation induces estrogen receptor α (ERα) expression. (a) Fli1 protein and mRNA levels and (b) ERα protein and mRNA levels after small interfering RNA (siRNA)-mediated Fli1 depletion in dermal fibroblasts. Cells were transfected with either 10nM of Fli1 siRNA or corresponding concentration of control siRNA. After 48hours, cells were serum-starved to remove the effect of serum. Cells were harvested 72hours after transfection. Protein levels of Fli1 and ERα were determined by western blot. The blots were reprobed with anti-β-actin antibody, and representative data of three independent experiments are shown. mRNA levels of Fli1 and ERα were determined by quantitative RT-PCR. Means±SD of three independent experiments is shown, with values normalized to control (arbitrarily set at 1). *P<0.01 (Student's t-test) versus control. Journal of Investigative Dermatology 2011 131, 1469-1476DOI: (10.1038/jid.2011.63) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Friend leukemia integration factor 1 (Fli1) occupies the estrogen receptor α (ERα) promoter. (a) ERα promoter (Kos et al., 2001). (b) ERα promoter utilization in dermal fibroblasts. Reverse transcription-PCR (RT-PCR) was performed using forward primers specific for each upstream exon and a reverse primer specific for exon 1. (c) ERα promoter A; Ets binding sites (closed ovals). (d) Chromatin immunoprecipitation (ChIP) assay of Fli1 occupancy of the promoter A. (e) Fli1 binding to the Ets sites in the ERα promoter using 5′ end-labeled wild-type (WT) and mutated at the Ets site (MUT) oligonucleotides. (f) Co-transfections of ERα-Luc with empty or Fli1 expression vector in fibroblasts with or without transforming growth factor-β (TGF-β) stimulation (Tb), normalized using pSV-β-galactosidase. The values depicted are means±SE of four experiments, *P<0.05. Journal of Investigative Dermatology 2011 131, 1469-1476DOI: (10.1038/jid.2011.63) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Estrogen receptor α (ERα) levels are elevated in Fli1+/− mouse fibroblasts and in the skin in vivo. Protein and mRNA levels of (a) friend leukemia integration factor 1 (Fli1) and (b) ERα in dermal fibroblasts from female Fli1+/+ and Fli1+/− mice. The value was normalized relative to that of Fli1+/+ mouse fibroblasts (arbitrarily set as 1) and means±SD of three experiments are shown. *P<0.05. (c) Immunodetection of ERα in skin samples from Fli1+/+ and Fli1+/− mice. Original magnification is 200-fold (upper panels) and 400-fold (lower panels). Scale bar=0.02mm. (b) The relative proportion of ERα-positive fibroblasts in dermis. At least 100 fibroblasts were counted for each specimen. The value was normalized relative to that of Fli1+/+ mouse skin (arbitrarily set as 1) and means±SD of three mice are shown. *P<0.05. Journal of Investigative Dermatology 2011 131, 1469-1476DOI: (10.1038/jid.2011.63) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Fli1+/− fibroblasts show enhanced fibrotic response to tamoxifen and 17β-estradiol stimulation. Dermal fibroblasts from (a) male and (b) female Fli1+/+ and Fli1+/− mice were stimulated for 24hours with 17β-estradiol (10−10M) (E2) or tamoxifen (10−7M) (TAM). Collagen α1(I) mRNA levels were determined by quantitative reverse transcription-PCR (qRT-PCR). Means±SD of three independent experiments is shown, with values normalized to control (arbitrarily set at 100). Representative western blot of collagen protein levels in the conditioned media is shown in the bottom panels. WT, wild type. Journal of Investigative Dermatology 2011 131, 1469-1476DOI: (10.1038/jid.2011.63) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions